Enzymes have well-defined three-dimensional structures formed by multiple noncovalent interactions such as hydrogen bonds, salt bridges, and hydrophobic interactions (Somero, 1995). At high temperatures, enzymes lose their original structure and denature to form insoluble aggregates that are no longer active (Somero, 1995; Rader et al., 2002; Fágáin, 1995). Because of their high efficiency and selectivity in catalyzing biological processes, enzymes are used for numerous industrial purposes (Rader et al., 2002; Ravindran and Son, 2011; Samejima et al., 1980; Schmid et al., 2001). However, this thermal instability of the proteins has negative impact on their applications in the pharmaceutical, food, and biotechnology industries. Many techniques such as chemical modification (DeSantis and Jones, 1999; Ryan et al., 1994) and protein engineering (Frosst et al., 1995; Matthews et al., 1987; Kumar et al., 2000; Imanaka et al., 1986) have been developed to address this problem. Additionally, polymers have been used as conjugates or excipients to enhance thermostability of enzymes (Gaertner and Puigserver, 1992; Longo and Combes, 1999; Yang et al., 1996; Kazan and Erarslan, 1997; Tomita et al., 2012). Yet some of these approaches are too expensive for certain industrial and agricultural applications.
For industrial applications, polymeric hydrogels are especially attractive materials for enzyme stabilization. Enzyme immobilization by hydrogels has been extensively studied in the context of industrial enzyme stabilization, especially to organic solvents (Sheldon, 2007). Enzymes can be loaded onto hydrogels without the need of a conjugation reaction, which simplifies the synthesis and stabilization process. And unlike polymer excipients that are difficult to remove from the enzyme solution, the macroscopic hydrogels can be easily separated by filtration or centrifugation. Due to these advantages, hydrogels have been frequently used for stabilization of enzymes as well as other proteins (Leobandung, 2002; Akiyoshi et al., 1999; Wang et al., 2008). Herein, we propose a novel hydrogel system based on trehalose as an effective excipient for enhancing the stability of enzymes at elevated temperatures.
Trehalose is a non-reducing disaccharide that has been shown to stabilize proteins and cells against stresses such as heat (Lippert and Galinski, 1992; Kaushik and Bhat, 2003; Baptista et al., 2008), desiccation (Guo et al., 2000; Hengherr et al., 2008; Crowe et al., 1984), and freezing (Beattie et al., 1997; Sundaramurthi and Suryanarayanan, 2009; Duong et al., 2006). Some animals accumulate trehalose to significant levels in response to environmental stresses (Westh and Ramlov, 1991; Madin and J. H. Crowe, 1975), emphasizing the ability of trehalose to stabilize biological molecules. Moreover, trehalose is generally regarded as safe (GRAS) (Jain and Roy, 2009) and is used in several pharmaceutical drugs as stabilizers (Ohtake and Wang, 2011). Our group has previously utilized trehalose-based linear polymers as excipients (Lee et al., 2013) or conjugates (Mancini et al., 2012) to stabilize proteins and retain their activity against heat and lyophilization. We sought to develop trehalose-based material to stabilize enzymes against heat and focused on hydrogels for the advantages described above.
Hydrogels have been extensively used as drug delivery vehicles with biomedical applications (Roy and Gupta, 2003). “Smart hydrogels”, which respond to specific triggers, can be synthesized to deliver and release guest drugs into a specifically targeted site (Bajpai et al., 2008; Gupta et al., 2002; Qiu and Park, 2001; Kiyonaka et al., 2002; Mano, 2008). In particular, pH responsive hydrogels are frequently used in drug delivery because different cell types and compartments of cells have discrete pHs, which allows for site specific release of a payload. For example, the pH of the extracelluar matrix (ECM) is typically around 7.4, while the cytosol has a lower pH and cancer cells are also more acidic than normal cells (Ingber et al., 1990; Wei et al., 2014). Moreover, the pH in the stomach is between pH 2 and 4 depending on whether the stomach is empty or food has been injested (Qiu and Park, 2001). Therefore research on pH responsive hydrogels is an important field of interest. Significant research has been reported toward the oral administration of therapeutics using pH responsive hydrogels. These hydrogels target the stomach for site-specific delivery of antibiotic, therapeutic proteins, and peptides (Lowman et al., 1999; Patel and Amiji, 1996; Besheer et al., 2006; Guo and Gao, 2007; Nho et al., 2005; Sajeesh and Sharma, 2006; Shantha and Harding, 2000). Since the target site is the stomach and stomach pH is 2-4 depending on empty or full, the hydrogels must only release their therapeutics in conditions more acidic than pH 4. This release occurs by changing the degree of swelling in the hydrogel or by cleaving the crosslinker.
Needed in the art are trehalose hydrogels for stabilization and delivery of proteins as animal feed stabilizers. Phytase is produced by bacteria found in the gut of ruminant animals (cattle, sheep) making it possible for them to use the phytic acid found in grains as a source of phosphorus. Non-ruminants (monogastric animals) like human beings, dogs, birds, etc. do not produce phytase. Research in the field of animal nutrition has put forth the idea of supplementing feed with phytase so as to make available to the animal phytate-bound nutrients like calcium, phosphorus, other minerals, carbohydrates and proteins.
This is a huge market with increasing importance for animal feed stabilizers (e.g., phytase). Needed in the art are trehalose-based hydrogels for stabilization and delivery of animal feed enzymes (e.g., phytase). These trehalose-based hydrogels should be responsive to the surrounded environments, e.g., pH values or the presence of glucose.
Insulin was the first Food and Drug Administration (FDA)-approved recombinant protein drug, and is widely used for the treatment of diabetes (Brown, 2005). However, one of the challenges associated with insulin therapy is the requirement of repeated injection or insertion of insulin bolus after each meal in the case of the insulin pump, which is problematic especially for children and young adults (Burdick et al., 2004). To address these challenges, phenylboronic acid that is non-toxic and durable has been widely used in materials for insulin release (Wu et al., 2011). Since boronic acid forms dynamic covalent complexes with 1,2- or 1,3-diols (Cambre and Sumerlin, 2011), its incorporation into hydrogels results in glucose-responsive materials. The two main mechanisms of insulin release from boronic acid hydrogels are swelling and competitive binding (Wu et al., 2011). The swelling mechanism is caused by the shift in the equilibrium of different boronic acid species toward the anionic tetrahedral form upon binding to diols such as those on sugars, which causes osmotic swelling of the hydrogels (Matsumoto et al., 2012). Alternatively, boronic acid-based polymers (Bapat et al., 2011) can form a hydrogel upon complexation with a diol-containing polymer in the presence of insulin, and later be competitively displaced by glucose to dissolve the hydrogel and release insulin (Wang et al., 2014).
In addition to controlled release of insulin, the instability of the protein is an important issue that needs to be addressed. Exposure of insulin to changes in temperature during storage may lead to inactivation of the protein resulting in health complications (Pryce, 2009). Instability also contributes to the medical costs of diabetes treatment because of protein that is discarded and wasted (Weiss et al., 2011). While insulin has been modified to increase its half-life in vivo (by covalent attachment of a polymer) (Hinds and Kim, 2002) and to prevent insulin hexamer formation (by mutation of the amino acid sequence) (Heise et al., 2007), only a few studies have reported stabilizing insulin to environmental heat exposure (Leobandung et al., 2002; Akiyoshi et al., 1998). Peppas has used nanospheres composed of poly(N-isopropylacrylamide) and poly(ethylene glycol) to enhance thermal and mechanical stability of insulin (Leobandung et al., 2002), but their system lacked a release mechanism. Akiyoshi et al. have used cholesterol-bearing pullulan nanogels to stabilize insulin against heat and enzymatic degradation, and the nanogel released insulin when exposed to physiological bovine serum albumin (BSA) level by association of BSA with pullulan (Akiyoshi et al., 1998). Although this system successfully stabilized insulin, it lacked glucose responsiveness, which is highly desirable in insulin delivery systems. To our knowledge, a hydrogel that is both glucose-responsive and insulin stabilizing has not yet been reported.
Needed in the art are trehalose hydrogels for stabilization and delivery of proteins. Needed in the art are trehalose-based hydrogels which are responsive to the surrounded environments, e.g., pH values or the presence of glucose.